Risk assessment for aortic dissection in Turner syndrome: The role of the aortic growth rate.

OBJECTIVE: The risk of aortic dissection (AoD) is increased in Turner syndrome (TS) but predicting those at risk is difficult. Based on scarce evidence, preventive aortic surgery is recommended when aortic diameter increases >5 mm/year. To investigate the aortic growth rate in TS and TS-related conditions associated with aortic growth. We also reported our experience of women who suffered aortic dissection (AoD), and who had preventive aortic replacement. METHODS: 151 adult TS were retrospectively identified. Women who had more than one transthoracic echocardiogram (TTE) after age 16 years were included in the aortic growth study. Aortic diameters at sinuses of Valsalva (SoV) and ascending aorta (AA) were analysed by two experts. RESULTS: 70/151 women had more than one TTE (interscan interval 4.7 years). Mean aortic growth was 0.13 ± 0.59 mm/year at SoV and 0.23 ± 0.82 mm/year at AA. Known risk factors for aortic dilatation and TS-related conditions were not associated with aortic growth. 4/151 women experienced AoD (age 25±8 years): two had paired scans for aortic growth, which was 0.67 mm/year at both SoV and AA in the first woman, and 11 mm/year (SoV) and 4 mm/year (AA) in the second. Only 1/4 of women with AoD survived; she used a TS cardiac-alert card to inform emergency personnel about her risk of AoD. 5/151 had a preventive aortic replacement, but one died post-operatively. CONCLUSIONS: Mean aortic growth in our TS population was increased compared to non-TS women and was not associated with currently known risk factors for AoD, suggesting that aortic growth rate itself could be a useful variable to stratify who is at risk for AoD.

Radiation biodosimetry: Current Status and Future Initiatives.

None.

Assessment of micronuclei frequency in the peripheral blood of adult and pediatric patients receiving fractionated total body irradiation.

The cytokinesis-block micronucleus (CBMN) assay is an established method for assessing chromosome damage in human peripheral blood lymphocytes resulting from exposure to genotoxic agents such as ionizing radiation. The objective of this study is to measure cytogenetic DNA damage and hematology parameters in vivo based on MN frequency in peripheral blood lymphocytes (PBLs) from adult and pediatric leukemia patients undergoing hematopoietic stem cell transplantation preceded by total body irradiation (TBI) as part of the conditioning regimen. CBMN assay cultures were prepared from fresh blood samples collected before and at 4- and 24- h after the start of TBI, corresponding to doses of 1.25 Gy and 3.75 Gy, respectively. For both age groups, there was a significant increase in MN yields with increasing dose (p < 0.05) and dose-dependent decrease in the nuclear division index (NDI; p < 0.0001). In the pre-radiotherapy samples, there was a significantly higher NDI measured in the pediatric cohort compared to the adult due to an increase in the percentage of tri- and quadra-nucleated cells scored. Complete blood counts with differential recorded before and after TBI at the 24 h time point, showed a rapid increase in neutrophil (p = 0.0001) and decrease in lymphocyte (p = 0.0006) counts, resulting in a highly elevated neutrophil-to-lymphocyte (NRL) ratio of 14.45 ± 1.85 after 3.75 Gy TBI (pre-exposure = 4.62 ± 0.49), indicating a strong systemic inflammatory response. Correlation of the hematological cell subset counts with cytogenetic damage, indicated that only the lymphocyte subset survival fraction (after-TBI compared with before-TBI) showed a negative correlation with increasing MN frequency from 0 to 1.25 Gy (r = -0.931; p = 0.007). Further, the data presented here indicate that the combination of CBMN assay endpoints (MN frequency and NDI values) and hematology parameters could be used to assess cytogenetic damage and early hematopoietic injury in the peripheral blood of leukemia patients, 24 h after TBI exposure.

Sex and dose rate effects in automated cytogenetics.

Testing and validation of biodosimetry assays is routinely performed using conventional dose rate irradiation platforms, at a dose rate of approximately 1 Gy/min. In contrast, the exposures from an improvised nuclear device will be delivered over a large range of dose rates with a prompt irradiation component, delivered in less than 1 µs, and a protracted component delivered over hours and days. We present preliminary data from a large demographic study we have undertaken for investigation of age, sex and dose rate effects on dicentric and micronucleus yields. Our data demonstrate reduced dicentric and micronucleus yields at very high dose rates. Additionally, we have seen small differences between males and females, with males having slightly fewer micronuclei and slightly more dicentrics than females, at high doses.

Analysis of a streamlined pathway for aortic surveillance for Turner syndrome in a single centre.

BACKGROUND AND OBJECTIVE: The risk of aortic dissection (AoD) is increased in women with Turner syndrome (TS) but predicting those with this heightened risk is difficult. In response to this, we sought to create a pathway to monitor TS patients to improve efficiency and resource utilisation in our dedicated TS clinic, and to monitor more closely those women thought to be at increased risk of AoD. DESIGN AND PATIENTS: Our pathway was designed based on evidence derived from International Guidelines for the management of aortic disease in women with TS. Women were divided according to those with known risk factors for AoD, and those with no known risk factors. These groups were further subdivided into 4 pathways depending on ascending aortic size which in-turn determined the frequency of outpatient appointments and imaging. RESULTS: Out of the 168 patients included in the analysis, 7 have had ascending aorta replacements, all in the highest risk group. Of the remaining 4 patients in the highest risk groups: 1 dissected whilst awaiting planned aortic surgery, 1 is currently awaiting surgery, 1 has low body mass index, therefore, making her aorta proportionally larger but not necessitating surgery and one has declined surgery. No women changed pathways. CONCLUSION: The risk-stratified pathway safely allowed consolidation of resources to women perceived to be at highest risk of AoD (excluding pregnancy), supporting the efficacy of the pathway and allowing the diversion of resources to those most at risk of AoD.

Development of a human peripheral blood ex vivo model for rapid protein biomarker detection and applications to radiation biodosimetry.

In the event of a widespread radiological incident, thousands of people may be exposed to a wide range of ionizing radiation. In this unfortunate scenario, there will be a need to quickly screen a large number of people to assess the amount of radiation exposure and triage for medical treatment. In our earlier work, we previously identified and validated a panel of radiosensitive protein biomarkers in blood leukocytes, using the humanized-mouse and non-human primate (NHP) models. The objective of this work was to develop a high-throughput imaging flow-cytometry (IFC) based assay for the rapid measurement of protein biomarker expression in human peripheral blood samples irradiated ex vivo. In this assay design, peripheral human blood samples from healthy adult donors were exposed to 0-5 Gy X-irradiation ex vivo and cultured for up to 2 days. Samples were stained with a cocktail of surface antigens (CD66b, CD20, and CD3), fixed and permeabilized, and intracellularly stained for BAX (Bcl-2-associated X) protein, used here as a representative biomarker. Samples were interrogated by IFC, and a uniform analysis template was created to measure biomarker expression in heterogeneous and specific leukocyte subtype populations at each time point. In this human blood ex vivo model, we show that within gated populations of leukocyte subtypes, B-cells are highly radiosensitive with the smallest surviving fraction, followed by T-cells and granulocytes. Dose-dependent biomarker responses were measured in the lymphocytes, B-, and T-cell populations, but not in the granulocytes, with dose-response curves showing increasing fold changes in BAX protein expression up to Day 2 in lymphocyte populations. We present here the successful use of this ex vivo model for the development of radiation dose-response curves of a candidate protein biomarker towards future applications of dose reconstruction and biodosimetry.

Comparison of isolated lymphocyte and whole blood based CBMN assays for radiation triage.

Following a mass-casualty nuclear/radiological event, there will be an important need for rapid and accurate estimation of absorbed dose for biological triage. The cytokinesis-block micronucleus (CBMN) assay is an established and validated cytogenetic biomarker used to assess DNA damage in irradiated peripheral blood lymphocytes. Here, we describe an intercomparison experiment between two biodosimetry laboratories, located at Columbia University (CU) and Health Canada (HC) that performed different variants of the human blood CBMN assay to reconstruct dose in human blood, with CU performing the assay on isolated lymphocytes and using semi-automated scoring whereas HC used the more conventional whole blood assay. Although the micronucleus yields varied significantly between the two assays, the predicted doses closely matched up to 4 Gy - the range from which the HC calibration curve was previously established. These results highlight the importance of a robust calibration curve(s) across a wide age range that match the exposure scenario as closely as possible and that will account for differences in methodology between laboratories. We have seen that at low doses, variability in the results may be attributed to variation in the processing whilst at higher doses the variation is dominated by inter-individual variation in cell proliferation. This inter-laboratory collaboration further highlights the usefulness of the CBMN endpoint to accurately reconstruct absorbed dose in human blood after ionizing radiation exposure.

Application of the cytokinesis-block micronucleus assay for high dose exposures using imaging flow cytometry.

The cytokinesis-block micronucleus (CBMN) assay is a well-established method to assess radiation-induced genetic damage in human cells. This assay has been adapted to imaging flow cytometry (IFC), allowing automated analysis of many cells, and eliminating the need to create microscope slides. Furthermore, to improve the efficiency of assay performance, a small-volume method previously developed was employed. Irradiated human blood samples were cultured, stained, and analysed by IFC to produce images of the cells. Samples were run using both manual and 96-well plate automated acquisition. Multiple parameter-based image features were collected for each sample and the results were compared to confirm that these acquisition methods are functionally identical. This paper details the multi-parametric analysis developed, and the resulting calibration curves up to 10 Gy. The calibration curves were created using a quadratic random coefficient model with Poisson errors, as well as a logistic discriminant function. The curves were then validated with blinded, irradiated samples, using relative bias and relative mean square error. Overall, the accuracy of the dose estimates was adequate for triage dosimetry (within 1 Gy of the true dose) over 90% of the time for lower doses and about half the time for higher doses, with the lowest success rate between 5 and 6 Gy where the calibration curve reached its peak and there was the smallest change in MN/BNC with dose. This work describes the application of novel multi-parametric analysis that fits the calibration curves and allows dose estimates up to 10 Gy, which were previously limited to 4 Gy. Furthermore, it demonstrates that the results from samples acquired manually and with the autosampler are functionally similar.

ST6GAL1-mediated aberrant sialylation promotes prostate cancer progression.

Aberrant glycosylation is a universal feature of cancer cells, and cancer-associated glycans have been detected in virtually every cancer type. A common change in tumour cell glycosylation is an increase in α2,6 sialylation of N-glycans, a modification driven by the sialyltransferase ST6GAL1. ST6GAL1 is overexpressed in numerous cancer types, and sialylated glycans are fundamental for tumour growth, metastasis, immune evasion, and drug resistance, but the role of ST6GAL1 in prostate cancer is poorly understood. Here, we analyse matched cancer and normal tissue samples from 200 patients and verify that ST6GAL1 is upregulated in prostate cancer tissue. Using MALDI imaging mass spectrometry (MALDI-IMS), we identify larger branched α2,6 sialylated N-glycans that show specificity to prostate tumour tissue. We also monitored ST6GAL1 in plasma samples from >400 patients and reveal ST6GAL1 levels are significantly increased in the blood of men with prostate cancer. Using both in vitro and in vivo studies, we demonstrate that ST6GAL1 promotes prostate tumour growth and invasion. Our findings show ST6GAL1 introduces α2,6 sialylated N-glycans on prostate cancer cells and raise the possibility that prostate cancer cells can secrete active ST6GAL1 enzyme capable of remodelling glycans on the surface of other cells. Furthermore, we find α2,6 sialylated N-glycans expressed by prostate cancer cells can be targeted using the sialyltransferase inhibitor P-3FAX -Neu5Ac. Our study identifies an important role for ST6GAL1 and α2,6 sialylated N-glycans in prostate cancer progression and highlights the opportunity to inhibit abnormal sialylation for the development of new prostate cancer therapeutics. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Improved prediction of breast cancer risk based on phenotypic DNA damage repair capacity in peripheral blood B cells.

Background: Standard Breast Cancer (BC) risk prediction models based only on epidemiologic factors generally have quite poor performance, and there have been a number of risk scores proposed to improve them, such as AI-based mammographic information, polygenic risk scores and pathogenic variants. Even with these additions BC risk prediction performance is still at best moderate. In that decreased DNA repair capacity (DRC) is a major risk factor for development of cancer, we investigated the potential to improve BC risk prediction models by including a measured phenotypic DRC assay. Methods: Using blood samples from the Breast Cancer Family Registry we assessed the performance of phenotypic markers of DRC in 46 matched pairs of individuals, one from each pair with BC (with blood drawn before BC diagnosis) and the other from controls matched by age and time since blood draw. We assessed DRC in thawed cryopreserved peripheral blood mononuclear cells (PBMCs) by measuring γ-H2AX yields (a marker for DNA double-strand breaks) at multiple times from 1 to 20 hrs after a radiation challenge. The studies were performed using surface markers to discriminate between different PBMC subtypes. Results: The parameter Fres, the residual damage signal in PBMC B cells at 20 hrs post challenge, was the strongest predictor of breast cancer with an AUC (Area Under receiver-operator Curve) of 0.89 [95% Confidence Interval: 0.84-0.93] and a BC status prediction accuracy of 0.80. To illustrate the combined use of a phenotypic predictor with standard BC predictors, we combined Fres in B cells with age at blood draw, and found that the combination resulted in significantly greater BC predictive power (AUC of 0.97 [95% CI: 0.94-0.99]), an increase of 13 percentage points over age alone. Conclusions: If replicated in larger studies, these results suggest that inclusion of a fingerstick-based phenotypic DRC blood test has the potential to markedly improve BC risk prediction.

Regional delivery in graduate nursing programs for students living in rural communities.

Oregon has a lack of primary care providers in rural areas. To address this issue, employers have indicated they plan to hire greater numbers of advanced practice registered nurses (APRNs). Oregon Health & Science University (OHSU) School of Nursing (SoN) responded to this need by developing a statewide delivery model to educate APRN students in their communities. A performance improvement work group including practice faculty, statewide academic leaders, and staff created a project charter with scope of work, timelines, and outcomes with the goal of improving the systems supporting APRN education. An initial distance APRN education delivery model emerged from this effort and was refined over the following year. Strategies were implemented to address identified challenges using small cycles of change. The final model has three main principles: being learner-centered, equitable, and sustainable. The central outcome is graduating students committed to practicing in rural and urban underserved communities to meet workforce needs in Oregon.

High-Throughput γ-H2AX Assay Using Imaging Flow Cytometry.

The γ-H2AX assay is a sensitive and reliable method to evaluate radiation-induced DNA double-strand breaks. The conventional γ-H2AX assay detects individual nuclear foci manually, but is labor-intensive and time-consuming, and hence unsuitable for high-throughput screening in cases of large-scale radiation accidents. We have developed a high-throughput γ-H2AX assay using imaging flow cytometry. This method comprises (1) sample preparation from small volumes of blood in the Matrix™ 96-tube format, (2) automated image acquisition of cells stained with immunofluorescence-labeled γ-H2AX using ImageStream®X, and (3) quantification of γ-H2AX levels and batch processing using the Image Data Exploration and Analysis Software (IDEAS®). This enables the rapid analysis of γ-H2AX levels in several thousand of cells from a small volume of blood with accurate and reliable quantitative measurements for γ-H2AX foci and mean fluorescence levels. This high-throughput γ-H2AX assay could be a useful tool not only for radiation biodosimetry in mass casualty events, but also for large-scale molecular epidemiological studies and individualized radiotherapy.

Tumour occurrence in women with Turner syndrome: A narrative review and single-centre case series.

BACKGROUND: Population studies suggest cancer morbidity may be different in Turner syndrome (TS) compared to the background female population. However, significant variability is observed in cancer associations likely due to heterogeneity in patient cohorts. We explored the prevalence and patterns of cancer amongst a cohort of women with TS attending a dedicated TS clinic. METHODS: Retrospective analysis of the patient database was performed to identify TS women who developed cancer. Population data (available before 2015) from the National Cancer Registration and Analysis Service database were used for comparison. RESULTS: Out of 156 TS women, median age of 32 (range 18-73) years, 9 (5.8%) had a recorded cancer diagnosis. Types of cancers were, bilateral gonadoblastoma, type 1 gastric neuroendocrine tumour (NET), appendiceal-NET, gastrointestinal stromal tumour, plasma cell dyscrasia, synovial sarcoma, cervical cancer, medulloblastoma and aplastic anaemia. Median age at cancer diagnosis was 35 (range 7-58) years and two were detected incidentally. Five women had 45,X karyotype, three received growth hormone treatment and all except one received oestrogen replacement therapy. The cancer prevalence of the background age-matched female population was 4.4%. CONCLUSIONS: We confirm the previous observations that women with TS do not appear to be at overall increased risk of common malignancies. Our small cohort showed a spectrum of rare malignancies that are not typically associated with TS, except for a single patient with a gonadoblastoma. The slightly higher prevalence of cancer in our cohort might simply represent increased cancer prevalence in the background population, or might be related to small sample size and regular monitoring of these women due to TS per se.

Longitudinal multi-omic changes in the transcriptome and proteome of peripheral blood cells after a 4 Gy total body radiation dose to Rhesus macaques.

BACKGROUND: Non-human primates, such as Rhesus macaques, are a powerful model for studies of the cellular and physiological effects of radiation, development of radiation biodosimetry, and for understanding the impact of radiation on human health. Here, we study the effects of 4 Gy total body irradiation (TBI) at the molecular level out to 28 days and at the cytogenetic level out to 56 days after exposure. We combine the global transcriptomic and proteomic responses in peripheral whole blood to assess the impact of acute TBI exposure at extended times post irradiation. RESULTS: The overall mRNA response in the first week reflects a strong inflammatory reaction, infection response with neutrophil and platelet activation. At 1 week, cell cycle arrest and re-entry processes were enriched among mRNA changes, oncogene-induced senescence and MAPK signaling among the proteome changes. Influenza life cycle and infection pathways initiated earlier in mRNA and are reflected among the proteomic changes during the first week. Transcription factor proteins SRC, TGFß and NFATC2 were immediately induced at 1 day after irradiation with increased transcriptional activity as predicted by mRNA changes persisting up to 1 week. Cell counts revealed a mild / moderate hematopoietic acute radiation syndrome (H-ARS) reaction to irradiation with expected lymphopenia, neutropenia and thrombocytopenia that resolved within 30 days. Measurements of micronuclei per binucleated cell levels in cytokinesis-blocked T-lymphocytes remained high in the range 0.27-0.33 up to 28 days and declined to 0.1 by day 56. CONCLUSIONS: Overall, we show that the TBI 4 Gy dose in NHPs induces many cellular changes that persist up to 1 month after exposure, consistent with damage, death, and repopulation of blood cells.

Pain Management and Substance Use Disorders: A Position Statement.

ABSTRACT: The American Society for Pain Management Nursing and the International Nurses Society on Addictions hold the position that persons with co-occurring pain and substance use disorder have the right to be treated with dignity and respect and receive evidence-based, high-quality assessment and management for both conditions using an integrated, holistic, multidimensional approach. Nonopioid and nonpharmacological approaches to pain management are recommended. Opioids should not be withheld from anyone if necessary to treat pain, and a team-based approach, including pain and addiction specialists, should be utilized when possible. Pain management should include interventions aimed at minimizing the risk for relapse or escalation of problematic substance use and actively involve the person and their support persons in the plan of care. Institutions should establish policies and procedures that support this position statement.

Upregulation of GALNT7 in prostate cancer modifies O-glycosylation and promotes tumour growth.

Prostate cancer is the most common cancer in men and it is estimated that over 350,000 men worldwide die of prostate cancer every year. There remains an unmet clinical need to improve how clinically significant prostate cancer is diagnosed and develop new treatments for advanced disease. Aberrant glycosylation is a hallmark of cancer implicated in tumour growth, metastasis, and immune evasion. One of the key drivers of aberrant glycosylation is the dysregulated expression of glycosylation enzymes within the cancer cell. Here, we demonstrate using multiple independent clinical cohorts that the glycosyltransferase enzyme GALNT7 is upregulated in prostate cancer tissue. We show GALNT7 can identify men with prostate cancer, using urine and blood samples, with improved diagnostic accuracy than serum PSA alone. We also show that GALNT7 levels remain high in progression to castrate-resistant disease, and using in vitro and in vivo models, reveal that GALNT7 promotes prostate tumour growth. Mechanistically, GALNT7 can modify O-glycosylation in prostate cancer cells and correlates with cell cycle and immune signalling pathways. Our study provides a new biomarker to aid the diagnosis of clinically significant disease and cements GALNT7-mediated O-glycosylation as an important driver of prostate cancer progression.

High-throughput measurement of double strand break global repair phenotype in peripheral blood mononuclear cells after long-term cryopreservation.

Peripheral blood mononuclear cells (PBMCs) are a useful model for biochemical assays, particularly for etiological studies. We describe here a method for measuring DNA repair capacity (DRC) in archival cryogenically preserved PBMCs. To model DRC, we measured γ-H2AX repair kinetics in thawed PBMCs after irradiation with 3 Gy gamma rays. Time-dependent fluorescently labeled γ-H2AX levels were measured at five time points from 1 to 20 h, yielding an estimate of global DRC repair kinetics as well as a measure of unrepaired double strand breaks at 20 h. While γ-H2AX levels are traditionally measured by either microscopy or flow-cytometry, we developed a protocol for imaging flow cytometry (IFC) that combines the detailed information of microscopy with the statistical power of flow methods. The visual imaging component of the IFC allows for monitoring aspects such as cellular health and apoptosis as well as fluorescence localization of the γ-H2AX signal, which ensures the power and significance of this technique. Application of a machine-learning based image classification improved flow cytometry fluorescent measurements by identifying apoptotic cells unable to undergo DNA repair. We present here DRC repair parameters from 18 frozen archival PBMCs and 28 fresh blood samples collected from a demographically diverse cohort of women measured in a high-throughput IFC format. This thaw method and assay can be used alone or in conjunction with other assays to measure etiological phenotypes in cryogenic biobanks of PBMCs.

Machine learning approach for quantitative biodosimetry of partial-body or total-body radiation exposures by combining radiation-responsive biomarkers.

During a large-scale radiological event such as an improvised nuclear device detonation, many survivors will be shielded from radiation by environmental objects, and experience only partial-body irradiation (PBI), which has different consequences, compared with total-body irradiation (TBI). In this study, we tested the hypothesis that applying machine learning to a combination of radiation-responsive biomarkers (ACTN1, DDB2, FDXR) and B and T cell counts will quantify and distinguish between PBI and TBI exposures. Adult C57BL/6 mice of both sexes were exposed to 0, 2.0-2.5 or 5.0 Gy of half-body PBI or TBI. The random forest (RF) algorithm trained on ½ of the data reconstructed the radiation dose on the remaining testing portion of the data with mean absolute error of 0.749 Gy and reconstructed the product of dose and exposure status (defined as 1.0 × Dose for TBI and 0.5 × Dose for PBI) with MAE of 0.472 Gy. Among irradiated samples, PBI could be distinguished from TBI: ROC curve AUC = 0.944 (95% CI: 0.844-1.0). Mouse sex did not significantly affect dose reconstruction. These results support the hypothesis that combinations of protein biomarkers and blood cell counts can complement existing methods for biodosimetry of PBI and TBI exposures.

A machine learning method for improving the accuracy of radiation biodosimetry by combining data from the dicentric chromosomes and micronucleus assays.

A large-scale malicious or accidental radiological event can expose vast numbers of people to ionizing radiation. The dicentric chromosome (DCA) and cytokinesis-block micronucleus (CBMN) assays are well-established biodosimetry methods for estimating individual absorbed doses after radiation exposure. Here we used machine learning (ML) to test the hypothesis that combining automated DCA and CBMN assays will improve dose reconstruction accuracy, compared with using either cytogenetic assay alone. We analyzed 1349 blood sample aliquots from 155 donors of different ages (3-69 years) and sexes (49.1% males), ex vivo irradiated with 0-8 Gy at dose rates from 0.08 Gy/day to ≥ 600 Gy/s. We compared the performances of several state-of-the-art ensemble ML methods and found that random forest generated the best results, with R2 for actual vs. reconstructed doses on a testing data subset = 0.845, and mean absolute error = 0.628 Gy. The most important predictor variables were CBMN and DCA frequencies, and age. Removing CBMN or DCA data from the model significantly increased squared errors on testing data (p-values 3.4 × 10-8 and 1.1 × 10-6, respectively). These findings demonstrate the promising potential of combining CBMN and DCA assay data to reconstruct radiation doses in realistic scenarios of heterogeneous populations exposed to a mass-casualty radiological event.